In this proposed study, we will test the hypothesis that the degradation of proteins by the ubiquitin-dependent pathway requires the intermediate formation of a unique multiubiquitin chain on a substrate protein. In this multiubiquitin chain, the C-terminal carboxyl group of one ubiquitin molecule is linked via an isopeptide bond to the e-amino group of lysine 48 of an adjoining ubiquitin molecule. Whether the degradation of a specific protein in the in vitro reticulocyte lysate system requires such a multiubiquitin chain intermediate can be tested initially by replacing wild-type ubiquitin in the degradative assay with a ubiquitin mutant, Ub-R48, in which lysine 48 was replaced by an arginine. Further structural studies can then be made to confirm the existence of this unique multiubiquitin chain in the degradative intermediates. The expression of Ub-R48 in the yeast Saccharomyces cerevisiae will also permit the testing of this hypothesis in vivo. In order to test the function of this multiubiquitin chain, we have also proposed to synthesize chemically a sufficient amount of an analog so that we may study its interaction with a purified protease which has been shown to act preferentially on ubiquitinated proteins. Positive results from these proposed studies should elucidate the mechanism by which proteolytic targeting is achieved by the posttranslational addition of ubiquitin to proteins. We also propose to characterize systematically the posttranslational addition of ubiquitin to proteins in the yeast nucleus. These characterizations will include the identification of nuclear ubiquitin conjugation enzymes and their substrates. Long term objectives in this research will include the cloning of the substrate proteins and studies of their functions by gene deletion and mutations. As part of our continuing effort to understand our finding of ubiquitin in the neurofibrillary tangles of Alzheimer's brain, we wish to determine how an insufficient level of cellular ubiquitin may cause cell death. Because the ubiquitin system is highly conserved among eukaryotes, relevant biochemical changes caused by an insufficient level of ubiquitin can be determined using the yeast Saccharomyces cerevisiae. Studies are proposed here to characterize the biochemical changes in a yeast mutant in which the polyubiquitin gene had been deleted.